Excitatory amino acid transporters (EAATs) in the CNS maintain extracellular glutamate concentrations below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. Over the previous grant period our laboratory identified and characterized five distinct human glutamate carrier subtypes, EAATs1-5. Transport of substrates by these carriers is thermodynamically coupled to the co-transport of two to three sodium ions, one proton, and the countertransport of a potassium ion; thus, this process is electrogenic. However, the amount of charge moved when substrates are applied is greater than would be predicted from the flux of coupled ions and substrate: we have also shown that this current elicited during substrate application arises from an uncoupled anion flux. Taking advantage of a highly functional cysteine-less version of EAAT1, we will continue to identify the structural and topological features required for substrate transport and ion permeation using cysteine substitutions together with sulfhydryl-modifying reagents. Studies will now begin to emphasize the electrophysiological analyses of currents associated with EAAT1 mutants using two electrode voltage clamp techniques in Xenopus oocytes. In addition, we will use two bacterial homologs of the mammalian EAATs as models for determining the structure of glutamate transporters with a view towards obtaining higher resolution structural information. Using high-resolution cryo-electron microscopic techniques we will image the bacterial carriers in lipid bilayers to determine gross structural features, including possible oligomeric state. Fluorescence and electron paramagnetic resonance (EPR) spectroscopy experiments will be used to refine structural models and analyze translocation-related conformational changes in purified bacterial carriers. The importance of understanding the structure, function, and regulation of different excitatory amino acid transporters is underscored by clinical and experimental studies, which have implicated abnormal transmitter reaccummulation in degenerative disorders such as ALS, Huntington's disease, ischemia-induced neurotoxicity, and Alzheimer's dementia.